GB/T 35337-2017 in English
VALIDDetection and identification of Grapevine yellow speckle viroid
- Issued on:-
- Implemented on:2018-07-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
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《GB/T 35337-2017葡萄黄点类病毒检疫鉴定方法》由TC271（全国植物检疫标准化技术委员会）归口，主管部门为国家标准化管理委员会。
Introduction
GB/T 35337—2017 Standard Overview
Grape Yellow Spot Viroid Quarantine and Identification Methods (GB/T 35337—2017) is an important standard in my country's plant quarantine field, which mainly stipulates the detection and identification methods of grape yellow spot viroids.
Standard Development Background and Technology Evolution Analysis
The standard was issued in 2017, based on internationally accepted virus detection technology and combined with my country's actual situation. Its technical core includes three methods: RT-PCR, real-time fluorescence RT-PCR and dot hybridization, which are characterized by high sensitivity and strong specificity.
| Standard Dimensions | Features of GB/T 35337—2017 | Advantages in International Comparison |
|---|---|---|
| Detection Methods | Covering RT-PCR, real-time fluorescence RT-PCR and dot hybridization techniques | Aligned with international standards to ensure mutual recognition of test results |
| Reagent Requirements | Clear requirements for reagent purity and source | Ensure accuracy and repeatability of experimental data |
| Result Determination | Multi-dimensional Verification System (Sequence Alignment, Ct Value Analysis) | Improve detection specificity |
Basic Information on Grape Yellow Spot Viroid
GYSVd stands for Grapevine yellow speckleviroid, an RNA virus that harms grape crops. It is mainly spread through grafting, mechanical transmission and seeds.
Detailed explanation of detection and identification methods
General RT-PCR detection method
1. Reagent preparation: including buffers such as potassium dihydrogen phosphate and sodium acetate.
2. Primer design: forward primer GYSVdF, reverse primer GYSVdR.
3. Detection process: RNA extraction → RT-PCR amplification → agarose gel electrophoresis verification → sequencing confirmation.
Real-time fluorescence RT-PCR detection method
1. Reagent preparation: 2× Real-time fluorescence RT-PCR Master Mix.
2. Primers and probes: GYSVd-1-F, GYSVd-1-R, GYSVd-1-P, etc.
3. Detection process: RNA extraction → real-time fluorescence PCR amplification → Ct value analysis → result determination.
Dot hybridization detection method
1. Probe preparation: cDNA cloning and transcription based on GYSVd sequence.
2. Sample processing: RNA denaturation and fixation.
3. Detection process: spot membrane → pre-hybridization → hybridization → development → result observation.
Instrument and reagent requirements
- PCR instrument: used for amplification reaction.
- Real-time fluorescence PCR instrument: used for quantitative analysis.
- Dot hybridization equipment: including hybridization oven and development system.
Result determination and recording
Sample testing needs to be verified by combining multiple methods (RT-PCR, real-time fluorescence RT-PCR, dot hybridization). Positive results need to be confirmed by sequence alignment or Ct value analysis to ensure the accuracy of the data.
Implementation suggestions
- Laboratory conditions: equipped with relevant instruments, equipment and reagents to ensure that the testing environment meets the requirements.
- Staff training: regular technical training is carried out to improve the standardization of operations.
- Quality control: establish positive and negative control systems to ensure the reliability of test results.
- Data management: Improve the experimental record and data analysis system to facilitate tracking and review.
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